In the field of the diagnosis or treatment of disease, attempts have been made for a long time to cause a desired substance to act selectively in a target cell or tissue while remaining only therein.
Particularly, in the field of the treatment of tumors or the diagnosis or treatment of osteoarthritis and brain disease, many studies on the proteins or small molecules present specifically in target lesions have been conducted. As a result, many types of materials specifically present in target lesions have been identified while studies on the use thereof for therapeutic purposes have also been actively conducted. For example, it has appeared that, in prostate cancer, prostate specific antigen (PSA) is largely present, and in other tumor tissues, matrix metalloprotease (MMP) is more highly expressed in normal tissue. Thus, such identified materials have been used as targets in the research and treatment of disease. However, if materials which are used for the diagnosis and treatment of diseases do not act specifically in such targets, problems of side effects or low image quality, which are caused by the non-specific distribution of the materials, will arise. For this reason, development of formulations which remain or act only in targets has been requested.
Generally, only some small molecules can enter the cytoplasm or nucleus of live cells through the cell membrane at a very low ratio, whereas large molecules cannot enter cells. However, because most materials, which are prepared for therapeutic, preventive or diagnostic purposes and the effective amount of which should be delivered into cells, are large molecules or macromolecules, methods of delivering biologically active macromolecules into cells without damaging the cells both in vivo and ex vivo have been demanded.
As a result of studies conducted to satisfy this demand, protein transduction domains (PTDs) have been suggested, and among them, TAT protein which is the transcription factor of human immunodeficiency virus-1 (HIV-1) has been most frequently studied. It was found that the TAT protein is more effective in passing through the cell membrane when it is composed of amino acids 47 to 57 (YGRKKRRQRRR), on which positively charged amino acids are concentrated, compared to when it is in a full-length form consisting of 86 amino acids (Fawell, S. et al., Proc. Natl. Acad. Sci. USA, 91:664, 1994). Other examples of PTDs include a peptide having a sequence of amino acids 267 to 300 of the VP22 protein of Herpes Simplex Virus type 1 (HSV-1) (Elliott G. et al., Cell 88:223-233, 1997), a peptide having a sequence of amino acids 84 to 92 of the UL-56 protein of HSV-2 (GeneBank code:D1047[gi:221784]), and a peptide having a sequence of amino acids 339 to 355 of the Antennapedia (ANTP) protein of Drosophila sp. (Schwarze, S. R. et al., Trends. Pharmacol. Sci., 21:45, 2000). In addition, artificial peptides consisting of positively charged amino acids also showed the effect of delivering drugs (Laus, R. et al., Nature. Biotechnol., 18:1269, 2000).
Recently, the present inventors reported the preparation of a low-molecular-weight protamine (LMWP) and the cell-penetrating activity thereof, in which the low-molecular-weight protamine (LMWP) has a peptide sequence similar to TAT, serves as a protein transduction domain and contains a large amount of cationic amino acids such as arginine. Particularly, the LMWP is a naturally occurring cationic peptide from protamine and is advantageous in that it presents no toxicity concerns and can be produced in large amounts (Park, Y. J. et al., J. Gene. Med., 700, 2003). However, this peptide is required to be additionally provided with target selectivity so that it is used for selective imaging, diagnostic or therapeutic purposes.
Accordingly, the present inventors have extensively made efforts to develop a method which can minimize problems of side effects or low image quality, attributable to the non-specific distribution of existing agents for the diagnosis or treatment of diseases, and which can effectively deliver a diagnostic or disease-treating agent specifically into a target cell. As a result, the present inventors have prepared a target-activated cell/tissue-penetrating peptide comprising a protein transduction domain (PTD), a spacer having a cleavage site specific for a target tissue enzyme, and a masking domain ionically bonded to the protein transduction domain (PTD), and have found that, when the prepared peptide is used as a conjugate with a drug, the drug is introduced specifically into a target cell/tissue, thereby completing the present invention.